The invention is in the field of molecular biology. It relates, more particularly, to cloned genes and their use in diagnostic, therapeutic and related applications.
The isolation of a novel peptide from lizard venom that displays 52% identity to mammalian GLP-1 prompted studies examining the biological properties of this protein, designated exendin 4 (Eng. et al. (1992) J.Biol. Chem. 267, 7402-7405). Experiments employing synthetic exendin 4 provided evidence that this peptide shares similar biological activities with mammalian GLP-1. Exendin 4 and truncated GLP-1[7-36)-amide increased cAMP levels in guinea pig acinar cell preparations (Raufman et al., 1992, J.Biol. Chem. 267, 21432-21437) and exendin 4 was subsequently shown to bind the GLP-1 receptor, stimulate glucose-dependent insulin secretion, and increase both cAMP accumulation and insulin gene expression in cultured islet cell lines in vitro (Goke et al., 1993, J.Biol. Chem. 268, 19850-19855). While full length exendin 4(1-39) is a strong agonist of GLP-1, truncated exendin 4(9-39)NH3 is a competitive antagonist of both exendin 4 and GLP-1. (Raufman, 1996, Regulatory Peptides 61:1-18. The insulinotropic GLP-1-like properties of exendin suggest that this lizard peptide may also be useful for the treatment of patients with diabetes.
A polynucleotide coding for reptilian exendin has now been isolated and characterized. Surprisingly, the polynucleotide isolated encodes a proexendin pleiotropic peptide which encodes a novel 47 amino acid peptide fused to the amino terminus of exendin (the 47 amino acid ENTP). The first 23 amino acids of the ENTP form a consensus signal peptide sequence. At the fusion between the novel peptide and exendin is a consensus dipeptidyl peptidase cleavage site. Cleavage at this site will excise a 2mer consisting of residues 46 and 47, and exendin 4. Further, an additional glycine residue was found at the carboxyl terminus of the deduced exendin coding sequence. This additional glycine residue is presumably cleaved during post-translational processing and carboxyl terminal amidation of exendin 4.
Accordingly, the present invention provides a polynucleotide encoding a proexendin. In one embodiment, the invention provides polynucleotides encoding lizard exendin 4 peptide and a 47 amino acid exendin N-terminal peptide (47 amino acid ENTP). In a related aspect, the invention provides polynucleotides encoding a 47 amino acid ENTP. In yet a further related aspect, the invention provides a polynucleotides encoding a 23 amino acid secretory signal peptide and a 22 amino acid ENTP.
In aspects of the invention, proexendin polynucleotide or a fragment thereof are utilized for further gene cloning to identify structurally related polynucleotides and species homologs. In related aspects of the invention, anti-sense versions of proexendin encoding polynucleotide and fragments thereof are obtained and utilized to regulate proexendin expression. Accordingly, the invention also provides, in one of its aspects, polynucleotides which hybridize to the polynucleotides encoding lizard proexendin, and complements thereof. Preferably, such nucleotides hybridize under medium stringent conditions, and encode functionally equivalent gene products. In yet another aspect of the invention, there is provided polynucleotides which hybridize under stringent conditions to polynucleotides encoding lizard proexendin, and complements thereof.
In another of its aspects, the proexendin encoded peptides are provided as products of recombinant production in a cellular host. In related aspects, there are provided recombinant host cells that express proexendin cDNA, and expression constructs in which polynucleotide coding for the proexendin encoded peptides are linked to expression controls functional in the selected host cell.
In another of its aspects, substantially purified novel exendin N-terminal peptides, and substantially purified exendin 4 (1-40) are provided. In yet another of its aspects, the invention provides specific antibodies directed to the 45 amino acid or 22 amino acid ENTPs, for use for example in diagnosis of conditions wherein the level of proexendin is altered.